Neurons communicate with each other in a neuronal network by firing action potentials. These electrical signals are converted into a chemical signal in the synapses between the neurons. Molecules responsible for this action are called neurotransmitters. The neurotransmitter L-glutamate is one of the most important chemical messengers in the synapses of the central nervous system. Detection of glutamate allows to monitor neuronal activity, which is a desirable tool e.g. for neurological research into neurodegenerative diseases, screening systems for neurotoxic compounds, screening of drugs that can influence synaptogenesis or synaptic activity. The detection of glutamate, a taste enhancer, also has applications in food industry.
Selective detection of glutamate can be done using glutamate agonists (e.g. AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)) or by enzymatic detection. The current on-chip enzymatic detection techniques rely on microelectrodes or ion-selective field-effect transistors (ISFET). Microelectrodes monitor the redox current upon catalysis of glutamate by the enzyme, while ion-sensitive field-effect transistors measure any change in local pH or charge at its liquid/surface interface.
Monitoring the chemical activity of neurons, requires a fast response, i.e. a sensor that quickly generates a detectable signal, in order to be able to monitor the activity in real time in the same time scale as the signal of the neurons. This speed relies not only on the rate of the chemical reaction that is selective for glutamate, but also on the swiftness of the sensor to pick up a detectable product of such chemical reaction and create a recognisable signal. Furthermore, due to the minute amount of neurotransmitters that is released by a neuron into the synaptic cleft, and the rapid diffusion of these molecules, a highly sensitive sensor is needed. For such purposes, field-effect transistors constitute prime candidates due to their fast response and their inherent, amplifying nature, and their ease to be miniaturised and integrated into large arrays with readout/drive electronics.
Different enzymes exist that selectively catalyse glutamate and generate a detectable (side) product.
Glutamate Dehydrogenase catalyses the reaction between L-glutamate, water and NAD+ to form 2-oxoglutarate and NADH. The side products of this reaction are ammonia and protons, and as such cause a change in pH. The enzyme, however, relies on exogenously added NAD+.
Glutamate Decarboxylase breaks down L-glutamate into 4-aminobutanoate and CO2. In water, CO2 generates carbonic acid, resulting in a decrease in pH. Glutamine Synthetase requires ATP and ammonium to convert L-glutamate into L-glutamine, and generates ADP and phosphate as side products. This approach has previously been reported (U.S. Pat. No. 4,812,220). The addition of exogenous ammonium as a substrate for the enzymatic reaction can be harmful for a neuron culture.
Glutamate Oxidase (GLOD) consumes only water and oxygen to convert L-glutamate into 2-oxoglutarate. The side products are ammonia and hydrogen peroxide, which cause a local change in pH.
In order to create a highly sensitive glutamate sensor, and as such accelerate its response, a lower limit of detection can be achieved by chemically amplifying the L-glutamate concentration, using a so-called “bienzymatic system” wherein the product of a first enzyme is the substrate of a second enzyme and vice versa. This chemical amplification principle has previously been demonstrated for the detection of lactate (Chaubey et al. (2001) Appl. Biochem. Biotechnol. 96, 239-248).
L-Glutamate Pyruvate Transaminase (GPT), can complement Glutamate Oxidase or Glutamate Dehydrogenase to form a bienzyme pair that amplifies the amount of L-glutamate produced by a cell. Glutamate Pyruvate Transaminase recycles 2-oxoglutarate from the first enzymatic reaction (with Glutamate Oxidase or Glutamate Dehydrogenase) and converts it back into L-glutamate (FIG. 2). The amino acid L-alanine is required to start and fuel this recycling reaction. Pyruvate is a side product of the reaction. As long as alanine is provided, a single molecule of L-glutamate cycles through this closed enzymatic loop and continues to generate ammonia and hydrogen peroxide as detectable products.
This bienzyme system is commercially available for the detection of Glutamate Oxidase in a solution [Molecular Probes, Invitrogen, the Netherlands]. Herein, the generation of H2O2 is used in a colorimetric reaction. Parton et al. (2005) in Solid State Technol. describe a glutamate sensor wherein a field effect transistor is covered with a layer of Glutamate Oxidase. This layer forms the contact between the electronic device and the neurons which adhere on top of this layer.
Castillo et al. (2005) Biosens. Bioelectron 20, 1559-1565, describe a sensor for the detection of glutamate wherein cells, growing on a porous membrane, are placed above an electrode comprising a hydrogel with Glutamate Oxidase. These authors emphasise that is it important to avoid contact of the cells with the electrode surface.